Genotyping of pathogenic leptospira by Multiple Locus Variable-number Tandem Repeat Analysis (MLVA)

ObjectiveTo introduce the method of molecular genotyping (MLVA) to determine the genotype of field isolates of leptospira.IntroductionLeptospirosis (ictherohemoglobinuria, Leptospirosis biliousness) is a natural focal and zoonotic infectious disease dangerous for humans and farm animals. It is important to identify specific leptospira strains isolated from rodents or sick and suspicious animals by the serotype or genotype. In comparison with serotyping using micro agglutination test (MAT), molecular genotyping makes it possible to accurately identify a specific pathogen strain. The genetic classification now becomes more significant than the phenotypic classification.MethodsSpecific oligonucleotide primers, which flank fragments of the genome locus of pathogenic leptospira varies in terms of the number of tandem repeats VNTR-4, -7, -10 specific for L.interrogans, L.kirschneri, and L.borgpetersenii were used. The amplification products were detected using agar gel electrophoresis with the following identification of the fragment length with a molecular weight marker and comparison with the collection of VNTR profiles of the strains described in the literature.ResultsIt was established that the method of leptospira molecular genotyping by determining the number of variable tandem repeats of a locus (VNTR-variable number tandem repeats analysis) is suitable for molecular epizootology studies in Ukraine. The advantages of the method are the simplicity of performance and availability for diagnostic and research laboratories in Ukraine compared to other pathogen genome sequencing based genotyping methods, in particular Multilocus sequence typing (MLST) or Multispacer Sequence Typing (MST), which require complex equipment and operating conditions. The reference strain of Leptospira M20 serotype Copengageni serogroup Icterohaemorrhagiae from the NAAS IVM collection of was studied and its VNTR profile was identified with the genotype of the strain Fiocruz L1-130 that is described in the literature as a serotype of Copengageni serogroup Icterohaemorrhagiae. The genotype of the leptospira field isolate obtained from a rat in Lviv Oblast of Ukraine was specified and its identity was established in the aforementioned genotype. The obtained data support the prospects of using MLVA genotyping method to study the distribution of different genotypes of leptospira. The research will continue to study the specificities of molecular epizootology of leptospirosis in Ukraine.ConclusionsThe method of leptospira molecular genotyping by multilocus analysis of the number of variable tandem repeats has been tested in the Leptospirosis Research Laboratory in collaboration with the Museum of Microorganisms at the National Academy of Sciences, the Ukraine Institute of Veterinary Medicine, the ELISA and PCR Research Laboratory, and the Bila Tserkva National Agrarian University. The genotype of the reference strain has been correlated with its serological profile; identification of the genotype of the field isolate pathogenic leptospira has been completed. The tested method is planned to be implemented in surveillance and control over leptospirosis spreading in Ukraine, and aimed to help in development and improvement of leptospirosis vaccine formulations. Additionally, method of Multiple-Locus Variable number tandem repeat Analysis will be used for molecular epidemiology research in Ukraine.ReferencesSalaün L, Mérien F, Gurianova S, Baranton G, Picardeau M. Application of multilocus variable-number tandem-repeat analysis for molecular typing of the agent of leptospirosis. J Clin Microbiol. 2006;44(11):3954-3962. doi:10.1128/JCM.00336-06.Caimi K, Repetto SA, Varni V, Ruybal P. Infection , Genetics and Evolution Leptospira species molecular epidemiology in the genomic era. Infect Genet Evol. 2017;54(July):478-485. doi:10.1016/j.meegid.2017.08.013.Ayral F, Zilber AL, Bicout DJ, Kodjo A, Artois M, Djelouadji Z. Distribution of leptospira interrogans by multispacer sequence typing in urban Norway rats (Rattus norvegicus): A survey in France in 2011-2013. PLoS One. 2015;10(10):1-14. doi:10.1371/journal.pone.0139604.

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Multilocus Variable Number of Tandem Repeat Analysis for molecular typing of uropathogenic Escherichia coli

10.21203/rs.3.rs-84882/v1 ◽

2020 ◽

Author(s):

Reza Ranjbar ◽

Farhad Safarpoor Dehkordi ◽

Morteza Mashhouri ◽

Omid Farahani

Keyword(s):

Escherichia Coli ◽

Tandem Repeat ◽

Tandem Repeats ◽

Genetic Relationships ◽

Variable Number Tandem Repeat ◽

Pcr Amplification ◽

Variable Number ◽

E Coli ◽

Repeat Analysis

Abstract Background: The aim of this study was genotyping of Uropathogenic Escherichia coli (UPEC) based on Variable Number of Tandem Repeats (VNTRs) sequences. Methods: E. coli strains isolated from urine samples were included in this study. Seven VNTR loci were subjected to Multilocus variable-number tandem repeat analysis (MLVA) based on PCR amplification. Then data was analyzed via online mlvaplus software and the information was displayed in the form of MST analysis. Results: A total of 100 E. coli strains were isolated and subjected to the study. MLVA was able to differentiate 56 different genotypes. Also, the technique could classify E. coli isolates in 5 clonal complexes. Based on UPGMA dendrograms, E. coli isolates were classified into 4 clusters (clusters A to D). The strains associated with Complex No. 1 appeared to be dominant pathogens of UPEC in Tehran's patients. The present study provides valuable insights into the genetic relationships of E. coli isolates recovered from clinical cases in a major hospital in Iran. Conclusions: The analysis of MLVA profiles using the MST algorithm showed the usefulness of the MLVA method in the classification of uropathogenic E. coli collected in different periods. We evaluated MLVA in a laboratory equipped with simple molecular equipment. Based on these results, it has been assumed that the E. coli strains were derived from a limited number of clones that have undergo a small genetic change during this period.

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Relatedness of Vibrio cholerae O1/O139 Isolates from Patients and Their Household Contacts, Determined by Multilocus Variable-Number Tandem-Repeat Analysis

Journal of Bacteriology ◽

10.1128/jb.00698-10 ◽

2010 ◽

Vol 192 (17) ◽

pp. 4367-4376 ◽

Cited By ~ 46

Author(s):

Emily A. Kendall ◽

Fahima Chowdhury ◽

Yasmin Begum ◽

Ashraful I. Khan ◽

Shan Li ◽

...

Keyword(s):

Vibrio Cholerae ◽

Tandem Repeat ◽

Tandem Repeats ◽

Genetic Relatedness ◽

Variable Number Tandem Repeat ◽

Index Patient ◽

Variable Number ◽

Repeat Analysis ◽

Household Contacts ◽

Vibrio Cholerae O1

ABSTRACT The genetic relatedness of Vibrio cholerae O1/O139 isolates obtained from 100 patients and 146 of their household contacts in Dhaka, Bangladesh, between 2002 and 2005 was assessed by multilocus variable-number tandem-repeat analysis. Isolate genotypes were analyzed at five loci containing tandem repeats. Across the population, as well as within households, isolates with identical genotypes were clustered in time. Isolates from individuals within the same household were more likely to have similar or identical genotypes than were isolates from different households, but even within a household, isolates from different individuals often had different genotypes. When household contacts were sampled regularly for 3 weeks after the illness of the household index patient, isolates with genotypes related to the index patient appeared in contacts, on average, ∼3 days after the index patient, while isolates with unrelated genotypes appeared in contacts ∼6 days after. Limited data revealed that multiple isolates from the same individual collected within days of each other or even from a single stool sample may have identical, similar, or unrelated genotypes as well. Our results demonstrate that genetically related V. cholerae strains cluster in local outbreaks but also suggest that multiple distinct strains of V. cholerae O1 may circulate simultaneously within a household.

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Comparison of Multilocus Variable-Number Tandem-Repeat Analysis and Whole-Genome Sequencing for Investigation of Clostridium difficile Transmission

Journal of Clinical Microbiology ◽

10.1128/jcm.01095-13 ◽

2013 ◽

Vol 51 (12) ◽

pp. 4141-4149 ◽

Cited By ~ 48

Author(s):

D. W. Eyre ◽

W. N. Fawley ◽

E. L. Best ◽

D. Griffiths ◽

N. E. Stoesser ◽

...

Keyword(s):

Clostridium Difficile ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Tandem Repeat ◽

Variable Number Tandem Repeat ◽

Variable Number ◽

Whole Genome ◽

Repeat Analysis

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Evaluating the Use of Multilocus Variable Number Tandem Repeat Analysis of Shiga Toxin-Producing Escherichia coli O157 as a Routine Public Health Tool in England

PLoS ONE ◽

10.1371/journal.pone.0085901 ◽

2014 ◽

Vol 9 (1) ◽

pp. e85901 ◽

Cited By ~ 17

Author(s):

Lisa Byrne ◽

Richard Elson ◽

Timothy J. Dallman ◽

Neil Perry ◽

Philip Ashton ◽

...

Keyword(s):

Public Health ◽

Escherichia Coli ◽

Tandem Repeat ◽

Shiga Toxin ◽

Variable Number Tandem Repeat ◽

Variable Number ◽

Escherichia Coli O157 ◽

Repeat Analysis

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Multilocus Variable-Number Tandem-Repeat Analysis, Pulsed-Field Gel Electrophoresis, and Antimicrobial Susceptibility Patterns in Discrimination of Sporadic and Outbreak-Related Strains of Yersinia enterocolitica

BMC Microbiology ◽

10.1186/1471-2180-11-42 ◽

2011 ◽

Vol 11 (1) ◽

pp. 42 ◽

Cited By ~ 30

Author(s):

Leila M Sihvonen ◽

Susanna Toivonen ◽

Kaisa Haukka ◽

Markku Kuusi ◽

Mikael Skurnik ◽

...

Keyword(s):

Yersinia Enterocolitica ◽

Tandem Repeat ◽

Antimicrobial Susceptibility ◽

Gel Electrophoresis ◽

Variable Number Tandem Repeat ◽

Variable Number ◽

Pulsed Field Gel Electrophoresis ◽

Pulsed Field ◽

Repeat Analysis ◽

Susceptibility Patterns

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Evaluation of Multiple-Locus Variable-Number Tandem-Repeat Analysis for Typing a Polyclonal Hospital-Acquired Methicillin-Resistant Staphylococcus aureus Population in an Area Where Such Infections Are Endemic

Journal of Clinical Microbiology ◽

10.1128/jcm.02220-09 ◽

2010 ◽

Vol 48 (8) ◽

pp. 2991-2994 ◽

Cited By ~ 8

Author(s):

B. Rivero-Perez ◽

E. Perez-Roth ◽

S. Mendez-Alvarez

Keyword(s):

Staphylococcus Aureus ◽

Tandem Repeat ◽

Methicillin Resistant Staphylococcus Aureus ◽

Variable Number Tandem Repeat ◽

Variable Number ◽

Methicillin Resistant ◽

Multiple Locus ◽

Repeat Analysis ◽

Hospital Acquired

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Multiple-Locus Variable-Number Tandem Repeat Analysis of Dutch Bordetella pertussis Strains Reveals Rapid Genetic Changes with Clonal Expansion during the Late 1990s

Journal of Bacteriology ◽

10.1128/jb.186.16.5496-5505.2004 ◽

2004 ◽

Vol 186 (16) ◽

pp. 5496-5505 ◽

Cited By ~ 99

Author(s):

Leo M. Schouls ◽

Han G. J. van der Heide ◽

Luc Vauterin ◽

Paul Vauterin ◽

Frits R. Mooi

Keyword(s):

The Netherlands ◽

Tandem Repeat ◽

Bordetella Pertussis ◽

Minimum Spanning Tree ◽

Whooping Cough ◽

Variable Number Tandem Repeat ◽

Clonal Expansion ◽

Variable Number ◽

Multiple Locus ◽

Repeat Analysis

ABSTRACT Bordetella pertussis, the causative agent of whooping cough, has remained endemic in The Netherlands despite extensive nationwide vaccination since 1953. In the 1990s, several epidemic periods have resulted in many cases of pertussis. We have proposed that strain variation has played a major role in the upsurges of this disease in The Netherlands. Therefore, molecular characterization of strains is important in identifying the causes of pertussis epidemiology. For this reason, we have developed a multiple-locus variable-number tandem repeat analysis (MLVA) typing system for B. pertussis. By combining the MLVA profile with the allelic profile based on multiple-antigen sequence typing, we were able to further differentiate strains. The relationships between the various genotypes were visualized by constructing a minimum spanning tree. MLVA of Dutch strains of B. pertussis revealed that the genotypes of the strains isolated in the prevaccination period were diverse and clearly distinct from the strains isolated in the 1990s. Furthermore, there was a decrease in diversity in the strains from the late 1990s, with a remarkable clonal expansion that coincided with the epidemic periods. Using this genotyping, we have been able to show that B. pertussis is much more dynamic than expected.

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